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Title: Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals

item Bannantine, John
item PAULSON, AVERY - University Of Nebraska
item CHACON, OFELIA - University Of Nebraska
item FENTON, ROBERT - University Of Nebraska
item ZINNIEL, DENISE - University Of Nebraska
item MCVEY, DAVID - University Of Nebraska
item SMITH, DAVID - University Of Nebraska
item CZUPRYNSKI, CHARLES - University Of Wisconsin
item BARETTA, RAUL - University Of Nebraska

Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2010
Publication Date: 1/1/2011
Citation: Bannantine, J.P., Paulson, A.L., Chacon, O., Fenton, R.J., Zinniel, D.K., Mcvey, D.S., Smith, D.R., Czuprynski, C.J., Baretta, R.G. 2011. Immunogenicity and reactivity of novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and conserved MAP1156 proteins with sera from experimentally and naturally infected animals. Clinical and Vaccine Immunology. 18(1):105-112.

Interpretive Summary: Our laboratory and collaborators at the University of Nebraska produced and genetically mapped transposon mutants of Mycobacterium avium subspecies paratuberculosis (MAP). One of these mutations results in phenotypic changes in colony morphology. In addition, this mutation cannot grow very well in bovine macrophages, which are cells designed to kill bacteria (attenuated mutation). However, MAP with no mutation is able to survive quite well in bovine macrophages. Therefore, this mutation is of interest as a potential vaccine. The mutation was defined in a gene cluster, one of which encodes a PPE protein. This is a family of virulence proteins that only occur in the genus Mycobacterium. The anitigenicity or immunoreactivity of this protein was determined. These studies show that the protein is immunogenic and the attenuated strain is a great vaccine candidate. The results of this study are of primary interest to scientists working in the field and also animal producers.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s Disease (JD) in ruminants. Development of genetic tools and completion of the MAP genome sequencing project expanded opportunities for antigen discovery. In this study, we determined the seroreactivity of two proteins encoded for at the 5’- and 3’- regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes for a PPE protein and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Maltose-binding protein (MBP) tagged recombinant MAP proteins were purified from Escherichia coli. Western immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of experimentally infected mice, rabbits; and naturally infected cattle. MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme linked immunosorbent assay for the recombinant proteins was developed and used to test pre-classified positive and negative serum samples from naturally infected and non-infected cattle. Samples, with one exception, displayed no seroreactivity against MBP-LacZ (P > 0.05), the negative control antigen. MAP1152 displayed seroreactivity against all positive sera, but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between non-infected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, MAP infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of immune-pathogenesis of JD.